The aim of this project was to investigate the feasibility of developing a PCR-based technique for analyzing genetic relatedness among isolates of Pneumocystis carinii from patients at the Clinical Center. Since this organism cannot be cultivated in vitro, the selective amplification of potentially variable portions of the genome directly from clinical specimens provides the only possibility for examining genetic variation. The genetic composition of P. carinii is poorly understood, and thus a limited number of targets are available for such exploration. We chose to examine intergenic spacer regions of the rRNA operon, a known source of intraspecific genetic variation in other microorganisms. The results of this study were very disappointing. Although we were able to amplify rRNA regions successfully from clinical specimens, we could find little evidence from exhaustive restriction endonuclease analysis for between- isolate variation in the sequence of the spacer region. Recently published data from laboratories employing similar approaches to us have shown that small differences in nucleotide sequence could be detected in this region by sequencing PCR products (a technique not well suited for clinical laboratories interested in typing P. carinii) and, that even when this highly sensitive method was employed, only 3-4 subtypes could be differentiated when a large number of isolates were examined. Such poor discriminatory power severely compromises the epidemiologic value of any data generated using this technique. Given these results, we have decided to discontinue this project until developments in P.carinii research make the possibility of developing a discriminatory typing system more feasible.